We wish to explore the role of cytochrome p450-dependent metabolites of arachidonic acid (AA) in the modulation of activity of apical K+ channels in the thick ascending limb (TAL) of the rat kidney. The apical K+ channels in the TAL play a key role in recycling K+ across the luminal membrane to provide an adequate supply of K+ for maintaining the normal function of the Na+/2Cl-/K+ cotransport. Cytochrome P450 metabolites of AA are important regulators of NaCl reabsorption in the TAL. The release of P450 metabolites of AA is enhanced by stimulation of phospholipase C and A2. Preliminary experiments have demonstrated that AA and 20 HETE, a major cytochrome p450~dependent metabolite of AA in the TAL, inhibit the apical K+ channels. Conversely, inhibition of cytochrome P450 increases the activity of the apical K+ channels. I propose to test the hypothesis that P450-dependent metabolites of AA are important for controlling the activity of the apical K channels and I will integrate this knowledge into the concepts of Na+Cl reabsorption in TAL. I plan to: 1) examine the specificity of the effects of AA and P450 metabolites of AA, 2) study the interaction of cytochrome P450 metabolites of AA with other intracellular second messengers, 3) explore the physiological importance of P450 metabolites of AA in interaction with hormones, such as vasopressin, angiotensin II (AII) and prostaglandin E2 (PGE2) in the modulation of the activity of the apical K+ channels. To achieve the first goal, patch-clamp techniques will be employed to study the effect of AA, metabolites of AA and other fatty acids on the activity of the apical K+ channels in the TAL. In addition, isolated perfused TAL tubules will be used to study the effect of AA and P450 metabolites on Na+ reabsorption. To achieve the second goal, I will study the effect of AA on channel activity in the presence of PKA or following inhibition of phosphatase, protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CamK II). In addition, the effect of AA will also be examined in a cloned renal K+ channel (ROMK) and its mutants (deletion of PKA or PKC sites). The following questions will be answered: 1) Are the effects of PKA, PKC and CaMK II modulated by AA? 2) Is the activity of phosphatase regulated by AA? 3) Is the sensitivity to AA preserved in the cloned renal K+ channel ? 4) which sites are required for the AA action? To achieve the third goal, the effect of vasopressin, AII and PGE2 on channel activity will be studied with or without inhibition of the cytochrome P450 metabolic pathway of AA. The experimental results may uncover a novel mechanism modulating the activity of the apical K+ channels as well as for the regulation of Na+Cl- reabsorption in the TAL.